The microscopic examination of tissue or tissue components is a common and valuable practice in both medicine and biology. Such procedures typically rely on the visual appearance of the tissue, which is often enhanced by the use of specialized stains that bind to certain tissue components, foreign bodies, or the products of cellular processes.
With the advent of computer technology, it has now become possible to automate many of the manual examination procedures by digitizing the images and placing them into the memory of a computer for analysis, display, and storage. However, the success of known automated imaging systems generally depends on the ability of the system to focus its optics on the tissue components of interest without operator intervention.
To obtain a focused image of a sample, microscopes have a stage for moving the objective or sample along the Z axis, thereby moving the focal plane of the objective towards or away from the sample; generally, either the objective or sample is in a fixed position and the other is moved along the Z axis. The Z axis is the axis perpendicular to a two-dimensional X,Y plane. Thus, where a sample is in an X,Y plane, such as on a generally planar slide, the Z axis is perpendicular to the slide extending from above and below the slide. A Z position is a position along the Z axis (e.g., position of focal plane, best focus, objective, stage, sample, or slide). There exists a focal plane located at a Z position which provides optimal focus of an image of the sample or portion thereof.
In order to assess the focus quality of a particular image, an image processor is typically used. The image processor quantifies the focusness of an image, so that an image with a peak of focus power can be identified. When an objective travels along the Z axis, a series of images are acquired. A focus power is calculated for each image acquired. Prior research and development has refined methods for calculating image focus power.
U.S. Patent Application No. 2004-0004614, entitled, Focusable Virtual Microscopy Apparatus and Method, describes a virtual microscope slide that includes images of a specimen for a given level of optical magnification, which are associated and stored in a data structure. The forming of the data structure that has the multiple Z axis images preferably includes automatically focusing at a principal reference focal plane and capturing and digitizing an optically magnified reference image and then shifting the specimen relative to the lens system by a predetermined increment to capture and digitize another Z axis image. Preferably, a multiple sequence of Z axis images above and below the reference image are captured and digitized.
While the '614 patent application provides a means of focusing on a particular X,Y coordinate, the '614 patent application may not provide an optimal focal plane. Rather, it builds a stack of images with focal planes at various Z axis locations, which allows the user to view each one. For an automated microscope imaging system, this utilizes considerable storage and processing resources and may not improve the speed at which the user can view images of magnified samples.
Patent Publication No. WO9704348A1, entitled, Automatic Focus System, describes an automated focus system formed by an intelligent, controlled electro-mechanical actuation system for maneuvering a microscope lens. The focus system brings a stained biological material into optimal focus for image acquisition. The automated focus system is operable without human intervention and utilizes a merit function based on the “texture” of a dark stained biological material in the field of view of the microscope objective. The system utilizes a maximization procedure that uses a feedback technique related to the merit function in order to control the position of the objective lens. The merit function, in turn, is based on a series of calculations performed on a set of digitized images captured at different focal positions. The use of an intelligent control routine to issue instructions to the motion control system allows the device to avoid the usual focus and pitfalls associated with microscopic image capture. The '348 patent publication describes a method, utilizing a Laplacian operator, to determine the focusness of a digital image taken of a sample with the focal plane at a particular location along the Z axis.
Once an image processor, using a focus algorithm, has quantified the focusness for several images taken at a particular X,Y location from several Z positions, the system then determines which image is most in focus. Current methods assume that the Z position with the greatest score is the image that is most in focus. However, if the system has focused on dust located on top of the cover slip, or if the specimen is translucent, there may be multiple peaks or no clear peak at all.
Once a current automated imaging system has determined optimal Z positions for each X,Y location, it fits a focal plane or surface to the resulting {X,Y,Z} coordinates. However, if the system focused on something other than the specimen at one or at a number of the focus points, the resulting focal plane may be significantly skewed towards these faulty points.